It is well known that sugars and other polyhydric compounds stabilize isolated proteins and phospholipid membranes during dehydration, probably by replacing the water molecules that are hydrogen-bonded to these macromolecules [reviewed by Crowe, J. H. et al. (1987) Biochemical Journal 242, 1-10]. Trehalose (.alpha.-glucopyranosyl-.alpha.-D-glucopyranose) is a dimer of two glucose molecules linked through their reducing groups. Because it has no reducing groups, it does not take part in the Maillard reactions that cause many sugars to damage proteins, and it is one of the most effective known protectants of proteins and biological membranes in vitro.
In nature, trehalose is found at high concentrations in yeasts and other fungi, some bacteria, insects, and some litoral animals, such as the brine shrimp. It is notable that all these organisms are frequently exposed to osmotic and dehydration stress. Accumulation of trehalose in higher plants is rare, but high levels occur in the so-called resurrection plants, such as the pteridophyte, Selaginella lepidophylla, which can survive extended drought [Quillet, M. and Soulet, M. (1964) Comptes Rendus de l'Academie des Sciences, Paris 259, pp. 635-637; reviewed by Avigad, G. (1982) in Encyclopedia of Plant Research (New Series) 13A, pp. 217-347].
A decreased availability of intracellular water to proteins and membranes is a common feature not only of dehydration and osmotic stress, but also of freezing, in which ice formation withdraws water from inside the cells, and heat stress, which weakens the hydrogen bonds between water and biological macromolecules. In recent years several publications have shown a close connection between the trehalose content of yeast cells, especially of the species Saccharomyces cerevisiae, and their resistance to dehydration and osmotic, freezing and heat stresses. This work has lead to the concept [summarized by Wiemkem, A. (1990) Antonie van Leeuwenhoek 58, 209-217] that, whereas the main storage or reserve carbohydrate in yeast is glycogen, the prime physiological function of trehalose is as a protectant against these and other stresses, including starvation and even poisoning by copper, ethanol and hydrogen peroxide, which all stimulate trehalose accumulation [Attfield, P. V. (1987) Federation of European Biochemical Societies Letters 225, 259-263].
Thus, during growth of Saccharomyces cerevisiae on glucose, glycogen begins to accumulate about one generation before the glucose is exhausted, and begins to be steadily consumed as soon as all external carbon supplies are exhausted. In contrast, accumulation of trehalose (partly at the expense of glycogen) only begins after all the glucose has been consumed, and the trehalose level is then maintained until nearly all the glycogen has been consumed [Lillie, S. A. & Pringle, J. R. (1980) Journal of Bacteriology 143, 1384-1394]. The eventual consumption of trehalose is accompanied by a rapid decrease in the number of viable cells.
When trehalose levels in S. cerevisiae have been manipulated by varying the growth conditions or administering heat shocks, high positive correlations have been found between the trehalose content of the cells and their resistance to dehydration [Gadd, G. et al. (1987) Federation of European Microbiological Societies Microbiological Letters 48, 249-254], heat stress [Hottiger, T. et al., (1987) Federation of European Biochemical Societies Letters 220, 113-115] and freezing [Gelinas, P. et al. Applied and Environmental Microbiology 55, 2453-2459]. Also, strains of S. cerevisiae and other yeasts selected for resistance to osmotic stress [D'Amore, T. et al. (1991) Journal of Industrial Microbiology 7, 191-196] or high performance in frozen dough fermentation [Oda, Y. (1986) Applied and Environmental Microbiology 52, 941-943] were found to have unusually high trehalose contents. Furthermore, a mutation in the cyclic AMP signaling system of S. cerevisiae that causes constitutive high trehalose levels also causes constitutive thermotolerance, whereas another mutation in this system that prevents the usual rise in trehalose during heat shock also prevents the acquisition of thermotolerance [Hottiger, T. et al., (1989) Federation of European Biochemical Societies Letters 255, 431-434]. Thus, there is much evidence pointing to a connection between trehalose content and stress resistance in yeasts, especially S. cerevisiae. Similar findings have been made for several other fungi [e.g., Neves, M. J., Jorge, J. A., Francois, J. M. & Terenzi, H. F. (1991) Federation of European Biochemical Societies Letters 283, 19-22]. However, a causative relationship has not yet been demonstrated. Further, nearly all conditions that cause increases in the trehalose content of yeast also cause increases in the levels of the so-called heat shock proteins. The 1989 publication of Hottiger and colleagues, cited above, claims that canavanine does not cause an increase in either trehalose levels or thermotolerance, whereas this compound is reported to induce heat shock proteins.
Whether or not there is a causal relation between trehalose content and stress resistance, it has become general practice in the manufacture of baker's yeast to maximise the trehalose content of the yeast. Various maturation processes have been developed to achieve this aim, and they are of crucial importance in the manufacture of active dried yeast. The details of these processes are often secret, but they are generally empirical regimes in which carbon and nitrogen feeds, aeration and temperature are carefully controlled and selected strains of yeast are used. They demand time and energy inputs during which little increase in cell number occurs. A more rational and controlled process would be of economic benefit.
According to Cabib, E. & Leloir, L. F. [(1957) Journal of Biological Chemistry 231, 259-275], trehalose is synthesized in yeast from uridine diphosphoglucose (UDPG) and glucose-6-phosphate (G6P) by the sequential action of two enzyme activities, trehalose-6-phosphate synthase (Tre6P synthase) and trehalose-6-phosphate phosphatase (Tre6Pase). Londesborough, J. & Vuorio, O. [(1991) Journal of Microbiology 137, 323-330, expressly incorporated herein by reference] have purified from baker's yeast a proteolytically modified protein complex that exhibited both these activities and appeared to contain a short polypeptide chain (57 kDa) and two truncated versions (86 kDa and 93 kDa) of a long polypeptide chain. The intact long chain was estimated to have a mass of at least 115 kDa. It was not disclosed which enzyme activity or activities was associated with which polypeptide, nor indeed whether both polypeptides were essential for either or both enzymatic activities. Anti-sera against both polypeptides were reported, but no amino acid sequences were disclosed.
An earlier patent application [EP 451 896; see claim 1] has claimed for a transformed yeast "comprising . . . one gene encoding . . . trehalose-6-phosphate synthase". However, no information about either the gene or the protein it encodes was provided.
Several authors have reported increases in Tre6P synthase activity in conditions that lead to accumulation of trehalose by S. cerevisiae, and Schizosaccharomyces pombe both during the approach to stationary phase [Winkler, K., et al. (1991) Federation of European Biochemical Societies Letters 291, 269-272; Francois, J., et al. (1991) Yeast 7, 575-587] and after temperature shift-ups to about 40.degree. C. [De Virgilio, C, et al. (1990) Federation of European Biochemical, Societies Letters 273, 107-110]. Panek and her colleagues [Panek, A. C., et al. (1987) Current Genetics 11, 459-465] have claimed that Tre6P synthase activity is increased by dephosphorylation of pre-existing enzyme molecules, i.e., that it is the result of post-translational regulation. This claim has been challenged [Vandercammen, A., et al., (1989) European Journal of Biochemistry 182, 613-620] but continues to be made [Panek, A. D. & Panek, A. C. (1990) Journal of Biotechnology 14, 229-238]. Evidence for or against an increase in the amount of enzyme during trehalose accumulation is conflicting. Inhibitors of mRNA synthesis inhibited trehalose accumulation by S. cerevisiae shifted from 30 to 45.degree. C. [Attfield (1987) loc.cit.], whereas under very similar conditions Winkler et al [(1991) loc.cit.] found that cycloheximide (an inhibitor of protein synthesis) did not prevent the accumulation of trehalose, which, however, occurred without an observable increase in Tre6P synthase activity. In a lower temperature range (a shift from 23 to 36.degree. C.), trehalose accumulation was accompanied by a three-fold increase in Tre6P synthase activity, and cycloheximide prevented the increase in Tre6P synthase [Panek, A. C., et al. (1990) Biochemie 72, 77-79]. In Schizosaccharomyces pombe, [De Virgilio, C., et al. (1991) loc. cit.] temperature shiftup caused a large accumulation of trehalose and increase of Tre6P synthase which were not prevented by cycloheximide, leading the authors to suggest that in this yeast a post-translational activation occurs. We now disclose that in S. cerevisiae the co-ordinate increases in Tre6P synthase and Tre6Pase activities during exhaustion of glucose are accompanied by an increase in antigenic material recognized by anti-sera to the short and long chains of a purified trehalose synthase. Hence, a method to increase the trehalose content of cells, and so, their stress tolerance, would be to isolate, clone, and modify the structural genes (hereinafter referred to as TPS1, TSL1, and TSL2) of these polypeptides and cause their expression in yeast or other host cells under the control of suitable promoters. If the expression of these genes could be controlled, then so could the trehalose content of the host cells.
The well known metabolic theory of Kacser & Burns [(1973) Symposium of the Society of Experimental Biology 27, 65-107] teaches that in principle the concentration of any intermediate, such as trehalose, can be increased by increasing the amount of any enzyme synthesizing it or decreasing the amount of any enzyme degrading it, but that the size of the increase may not be significant. The novelty of the present invention lies in the identification and characterization of the particular yeast genes that must be modified to increase the amounts of trehalose synthase and the recognition of the advantages of modifying the synthetic pathway rather than the degradative pathway. These advantages include (i) leaving the highly regulated [see, e.g., Thevelein, J. M. (1988) Experimental Mycology 12, 1-12] degradative pathway intact to avoid the physiological problems likely in yeast that cannot activate this pathway, (ii) the possibility of causing yeast to synthesize trehalose under physiological conditions where wild type yeasts do not (so that blocking the degradative pathway cannot increase the amount of trehalose) and (iii) the important possibility of introducing by these genes a trehalose-synthetic capacity to organisms, such as most higher plants, that naturally lack this capacity.
Expression of the genes for trehalose synthesis in yeast under conditions where trehalase is active will increase the operation of a so-called "futile" cycle, in which glucose is continuously phosphorylated, converted to trehalose and regenerated by hydrolysis of the trehalose, resulting in increased consumption of ATP. This ATP must be regenerated, and under fermentative conditions this will occur by conversion of sugars into ethanol. Therefore, introduction of TPS1, TSL1 and TSL2 into yeast under the control of promoters active under fermentative conditions is expected to decrease the yield of cell mass on carbon source and increase that of ethanol. The many attempts [e.g., Schaaf et al. (1989) Yeast 5, 285-290] to increase fermentation rates in yeast by increasing the levels of glycolytic enzymes have been unsuccessful. The probable reason is that availability of ADP limits the rate of glycolysis in yeast. Introduction of a futile cycle-ATPase is thus expected to increase this rate. The feasibility of this invention is demonstrated by the finding [Gancedo, J. -M. & Navas, M. A. (1992) Yeast 8, S574] that expression of the gluconeogenic enzymes, fructose bisphosphatase and phosphoenolpyruvate carboxykinase under fermentative conditions (so causing two futile cycles) caused a 50% increase in the fermentation rate of resting yeast. Use of the trehalose futile cycle has the added advantage that the cells must then contain a steady state level of trehalose, which increases their tolerance to osmotic and temperature stress.
The present invention includes transformed strains of distiller's yeast, in which the presence of modified forms of any or all of TPS1, TSL1 and TSL2 results in an increased yield of ethanol from carbohydrate sources.
As well as being used to improve the properties of yeast, especially active dried yeast and yeast for frozen doughs, this invention has other obvious applications. First, by increasing the proportion of trehalose in yeast, the industrial scale production of trehalose from yeast is made more economic. It is particularly advantageous to obtain trehalose from yeast because, since yeast is a traditional and safe food stuff, a minimal purification of the trehalose will often be adequate: preparations of trehalose containing yeast residues could be safely added to food stuffs for human or animal consumption. Trehalose also has medical applications, both as a stabilizer of diagnostic kits, viruses and other protein material [WO 87/00196] and, potentially, as a source of anti-tumour agents [Ohtsuro et al. (1991) Immunology 74, 497-503]. Trehalose for internal applications in humans would be much more safely obtained from yeast than from a genetically engineered bacterium.
Second, by transferring these genes to higher plants after making suitable modifications obvious to anyone skilled in the art (in general, replacements of adenine/thymine base pairs by guanine/cytosine base pairs as suggested by Perlak et al. [(1991) Proceedings of the National Academy of Sciences of the U.S.A. 88, 3324-3328] and the introduction of suitable promoters, some of which may be tissue-specific, to direct the synthesis of trehalose to frost and drought-sensitive tissues), the resistance of the plants to various stresses, especially frost and dehydration, should be improved. Other microbial trehalose synthases could also be used, including those of Candida utilis (Soler et al [1989] FEMS Microbiol Letters 61, 273-278), E. coli (Glaever et al [1988] J. Bacteriol. 170, 2841-2849), Dictyostelium discoideum (Killick [1979] Arch. Biochem. Biophys. 196, 121-133) and Mycobacterium smegmatis (Lapp et al [1971] J. Biol. Chem. 246, 4567-4579), the latter two systems being able to use ADPG as an alternative to UDPG. The economic importance of such improvements is potentially enormous, because even small increases in cold-tolerance will lead to large increases in growing season, whereas dehydration resistance can save entire crops in time of drought. Frost and drought resistance in higher plants is usually accompanied by increases in compounds such as proline rather than trehalose [reviewed by Stewart (1989) in "Plants under Stress", pp 115-130], but, as mentioned above, resurrection plants accumulate large amounts of trehalose and there seems, a priori, to be no reason why this strategy should not be successful. Therefore, the present invention includes a process to transform crop plants by introducing recombinant forms of the structural genes for microbial trehalose synthases (such as the TPS1, TSL1 and TSL2 genes for the yeast enzyme) so as to increase the trehalose content of some of their tissues compared to those of the untransformed plant. Such transformed plants can also be economic and safe sources of trehalose. Third, the shelf-life of food products can be increased by adding trehalose to them [WO 89/00012]. A further aspect of the present invention is a novel process for producing trehalose-enriched food products from plants by causing them to express the structural genes for a microbial trehalose synthase in their edible tissues.